Cells interact with their environments through membrane proteins. Structural and functional studies of membrane proteins are thus very important. Structure determination of eukaryotic membrane proteins in membrane however remains difficult despite substantial progresses. Part of the challenge comes from the fact that many eukaryotic membrane proteins undergo a complicated intracellular maturation process and carry different post-translational modifications before reaching their final destinations. Current high throughput crystallization and cryoEM single particle reconstruction are largely carried out with proteins in detergents, or in membrane-mimetic systems such as bicelles, nanodiscs, lipid-cubic phases or amphipols, where there are still significant differences in comparison with a native membrane. New technologies are needed to overcome these problems. We propose here to develop two new technologies for cryoEM study of membranes proteins in continuous membrane using type 1 IP3 receptor (IP3R) as a working model. The premise of these two methods is partly based on our recent work of a chemical engineering procedure that is suitable for functionalizing nanometer-thick carbon films and of a bead-supported spherical unilamellar membrane (bSUM) system that allows the generation of stable giant unilamellar vesicles. With milligram amounts of IP3R proteins, we will produce a nanometer-bSUM (nm-bSUM) and a carbon-supported planar unilamellar membrane (cPUM). These two systems will be prepared for the cryoEM visualization of the IP3Rs in continuous membrane where the proteins are fully immersed in a lipid bilayer, and will allow us to resolve the receptor structure from images of membrane-integrated molecules. Images of the receptors in nm-bSUMs will be used for random spherically constrained (RSC) reconstruction. Receptors in cPUMs will be imaged at high tilt angles for 3D reconstruction with corrections for changes in defocus levels across the imaging field. Both methods will rely on chemical engineering and membrane reconstitution at the nanometer scale and will result in efficient unidirectional insertion of membrane proteins at sub-nM concentrations, which will be particularly beneficial for selecting specifically labeled mature functional membrane proteins or enriching low-abundance membrane protein complexes at sub-nM concentrations. Results of the proposed studies will create new windows of opportunities for cryo-EM study of various membrane protein complexes in membrane and for using nanoscale membrane systems in other bioanalytical or biomedical applications. ! 1!